The subject matter of the present invention is directed to a stable, therapeutically effective composition containing cell wall skeleton (CWS) and purified trehalose dimycolates (TDM). Both substances are isolates of bacteria and when used together as a composition, are effective in obtaining suppression and regression of tumor cells. The present invention also includes a method of preparing the composition as well as use of the composition in treating cancerous tumors and as an adjuvant.
The combination of CWS and TDM is known in the art (See Biologically Active Components from Mycobacterial Cell Walls. II. Suppression and Regression of Strain-2 Guinea Pig Hepatoma; Meyer et al., Journal of the National Cancer Institute, Volume 52, No. 1, January, 1974; and Mycobacterial Cell Wall Components in Tumor Suppression and Regression; Ribi et al., National Cancer Institute Monograph No. 39, pgs. 115-120, October, 1972) incorporated herein by reference.
Cell Wall Skeleton is essentially cell wall which has had much of the protein and lipids normally found in the cell wall removed. It is a polymeric mycolic acid--arabinogalactan mucopeptide containing remnants of trehalose mycolates ("P.sub.3 ") and undigested tuberculoproteins. Cell wall skeleton is obtained from any mycobacteria including, but not limited to, M.smegmatis, M.phlei, M.avium, Nocardia rubra, Nocardia asteroides, Corynebacterium diphtheriae, Corynebacterium parvum, M.bovinis, M.kansasii, M. tuberculosis (Strain H 37 RV and Ayoma B), and M.Bovis Strain BCG. Additionally, cell wall skeleton may be obtained from such non-mycobacteria as E.coli, B.abortus and Coxiella burnettii.
The process of producing cell wall skeleton is time consuming. The bacteria such as M.Bovis Strain BCG (Bacillus Calmette-Guerin) is grown and harvested. The resulting whole cell residue is processed through a cell fractionator [Ribi Cell Fractionator (Sorvall, Model RF-1)] which disrupts the cells, separating the outer envelope or cell wall from the protoplasmic impurities. The resulting cell walls are then subjected to a series of solvent extractions and enzymatic treatments (e.g., trypsin and/or chymotrypsin) to give purified cell wall skeleton.
The second component of the instant composition, trehalose dimycolates (TDM), may be obtained from any mycobacteria as, for example, M.avium, M.phlei, M.tuberculosis (Strain H 37 RV and Ayoma B), M.bovis BCG, M.smegmatis, M.kansasii, Nocardia rubra, M.bovinis and Corynebacterium diphtheriae.
Bacteria such as M.avium is grown, harvested and then heat killed. The cell mass is then extracted with several solvents and then an active, solvent soluble, fraction is extracted. This extract is further purified by a series of solvent extractions to provide crude TDM (See Biologically Active Components from Mycobacterial Cell Walls. I. Isolation and Composition of Cell Wall Skeleton and Component P.sub.3 ; Azuma, et al., Journal of the National Cancer Institute, Volume 52, pgs. 95-101, 1974) incorporated herein by reference. As disclosed in Azuma et al, crude TDM may then be further purified by centrifugal microparticulate silica gel chromatography to give purified TDM.
CWS and TDM produced as described above have been combined in an oil droplet emulsion. The non-living components are ground with a small amount of mineral oil and emulsified in saline to produce an anti-animal tumors composition suitable for injection (See Immunotherapy with Non-viable Microbial Components, Ribi, et al.; Annals of the National Academy of Sciences, Volume 227, pgs. 228-238, Sept. 20th, 1976) incorporated herein by reference.
However, the prior art oil in saline emulsions containing CWS and TDM suffer from a major disadvantage. The emulsion has a relatively short shelf life at room temperature and, therefore, must be used shortly after preparation to produce the desired results.
It is well-known in the art that lyophilizing a pharmaceutical preparation can extend shelf life considerably (See, for example, U.S. Pat. No. 3,932,943; U.S. Pat. No. 3,594,471; and U.S. Pat. No. 4,134,214). To be successful, the lyophilized product must be able to be reconstituted at a later time without any loss in potency, that is, with the same potency as the pre-lyophilized product. However, the prior art CWS-TDM oil in saline emulsions have not been effectively lyophilized. Applicants have discovered that if these emulsions are not stabilized without delay, as, for example, by lyophilization, they will begin a process of degradation resulting in a significant percent of oil droplets becoming uncoated. The uncoated material is not active in tumor regression. The therapeutically effective emulsion of the present invention is stabilized by a lyophilization procedure; other stabilization procedures can also be utilized.
It is therefore an object of the invention to provide a stable CWS-TDM composition which may be effectively reconstituted to produce an effective anti-animal tumors preparation having a superior shelf life that is, a shelf life of a year and even longer. It is another object of the invention to provide a CWS-TDM composition having a large numer of coated oil droplets which are very effective in tumor regression, without side effects.
It is still another object of the invention to provide a method of treating various cancer tumors with a stable and potent CWS-TDM composition. It is another object of the invention to employ the CWS-TDM composition as an adjuvant as for example, to increase the immune response to immunogens including, for example, microorganisms, proteins, carbohydrates, allergens, viruses and the like.